Targets in the Tumour Matrisome to Promote Cancer Therapy Response.

The extracellular matrix (ECM) is composed of complex fibrillar proteins, proteoglycans, and macromolecules, generated by stromal, immune, and cancer cells. The components and organisation of the matrix evolves as tumours progress to invasive disease and metastasis. In many solid tumours, dense fibrotic ECM has been hypothesised to impede therapy response by limiting drug and immune cell access. Interventions to target individual components of the ECM, collectively termed the matrisome, have, however, revealed complex tumour-suppressor, tumour-promoter, and immune-modulatory functions, which have complicated clinical translation. The degree to which distinct components of the matrisome can dictate tumour phenotypes and response to therapy is the subject of intense study. A primary aim is to identify therapeutic opportunities within the matrisome, which might support a better response to existing therapies. Many matrix signatures have been developed which can predict prognosis, immune cell content, and immunotherapy responses. In this review, we will examine key components of the matrisome which have been associated with advanced tumours and therapy resistance. We have primarily focussed here on targeting matrisome components, rather than specific cell types, although several examples are described where cells of origin can dramatically affect tumour roles for matrix components. As we unravel the complex biochemical, biophysical, and intracellular transduction mechanisms associated with the ECM, numerous therapeutic opportunities will be identified to modify tumour progression and therapy response.

The Future of Precision Oncology.

Our understanding of the molecular mechanisms underlying cancer development and evolution have evolved rapidly over recent years, and the variation from one patient to another is now widely recognized. Consequently, one-size-fits-all approaches to the treatment of cancer have been superseded by precision medicines that target specific disease characteristics, promising maximum clinical efficacy, minimal safety concerns, and reduced economic burden. While precision oncology has been very successful in the treatment of some tumors with specific characteristics, a large number of patients do not yet have access to precision medicines for their disease. The success of next-generation precision oncology depends on the discovery of new actionable disease characteristics, rapid, accurate, and comprehensive diagnosis of complex phenotypes within each patient, novel clinical trial designs with improved response rates, and worldwide access to novel targeted anticancer therapies for all patients. This review outlines some of the current technological trends, and highlights some of the complex multidisciplinary efforts that are underway to ensure that many more patients with cancer will be able to benefit from precision oncology in the near future.

PKN2 deficiency leads both to prenatal 'congenital' cardiomyopathy and defective angiotensin II stress responses.

The protein kinase PKN2 is required for embryonic development and PKN2 knockout mice die as a result of failure in the expansion of mesoderm, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The specific role of PKN2 in contractile cardiomyocytes during development and its role in the adult heart remain to be fully established. We used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency to assess cardiac development and function using high resolution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were also assessed. Cardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myocardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed up-regulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardiomyocyte-specific deletion of PKN2, the requirement for PKN2 in adult mice was explored using the constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in these haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis. It is concluded that cardiomyocyte PKN2 is essential for heart development and the formation of compact myocardium and is also required for cardiac hypertrophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.

Cancer-associated fibroblasts in pancreatic cancer: new subtypes, new markers, new targets.

Cancer-associated fibroblasts (CAFs) have conflicting roles in the suppression and promotion of cancer. Current research focuses on targeting the undesirable properties of CAFs, while attempting to maintain tumour-suppressive roles. CAFs have been widely associated with primary or secondary therapeutic resistance, and strategies to modify CAF function have therefore largely focussed on their combination with existing therapies. Despite significant progress in preclinical studies, clinical translation of CAF targeted therapies has achieved limited success. Here we will review our emerging understanding of heterogeneous CAF populations in tumour biology and use examples from pancreatic ductal adenocarcinoma to explore why successful clinical targeting of protumourigenic CAF functions remains elusive. Single-cell technologies have allowed the identification of CAF subtypes with a differential impact on prognosis and response to therapy, but currently without clear consensus. Identification and pharmacological targeting of CAF subtypes associated with immunotherapy response offers new hope to expand clinical options for pancreatic cancer. Various CAF subtype markers may represent biomarkers for patient stratification, to obtain enhanced response with existing and emerging combinatorial therapeutic strategies. Thus, CAF subtyping is the next frontier in understanding and exploiting the tumour microenvironment for therapeutic benefit. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Targeting the HER3 pseudokinase domain with small molecule inhibitors.

HER3 is a potent oncogenic growth factor receptor belonging to the human epidermal growth factor (HER/EGFR) family of receptor tyrosine kinases. In contrast to other EGFR family members, HER3 is a pseudokinase, lacking functional kinase activity. As such, efforts to develop small molecule tyrosine kinase inhibitors against this family member have been limited. In response to HER3-specific growth factors such as neuregulin (NRG, also known as heregulin or HRG), HER3 must couple with catalytically active family members, including its preferred partner HER2. Dimerization of the intracellular HER2:HER3 kinase domains is a critical part of the activation mechanism and HER3 plays a specialized role as an allosteric activator of the active HER2 kinase partner. Intriguingly, many pseudokinases retain functionally important nucleotide binding capacity, despite loss of kinase activity. We demonstrated that occupation of the nucleotide pocket of the pseudokinase HER3 retains functional importance for growth factor signaling through oncogenic HER2:HER3 heterodimers. Mutation of the HER3 nucleotide pocket both disrupts signaling and disrupts HER2:HER3 dimerization. Conversely, ATP competitive drugs which bind to HER3, but not HER2, can stabilize HER2:HER3 dimers, induce signaling and promote cell growth in breast cancer models. This indicates a nucleotide-dependent conformational role for the HER3 kinase domain. Critically, our recent proof-of-concept work demonstrated that HER3-directed small molecule inhibitors can also disrupt HER2:HER3 dimerization and signaling, supporting the prospect that HER3 can be a direct drug target despite its lack of intrinsic activity. In this chapter we will describe methods for identifying and validating small molecule inhibitors against the HER3 pseudokinase.

Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion.

In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

Uncoupling TORC2 from AGC kinases inhibits tumour growth.

Mammalian target of rapamycin (mTOR) is a central regulator of growth and metabolism. mTOR resides in two distinct multi-protein complexes - mTORC1 and mTORC2 - with distinct upstream regulators and downstream targets. While it is possible to specifically inhibit mTORC1 with rapamycin, or inhibit both mTOR complexes together with ATP pocket directed mTOR kinase inhibitors, it is not possible to assess the specific roles for mTORC2 pharmacologically. To overcome this, we have developed a novel, inducible, dominant negative system for disrupting substrate recruitment to mTORC2. Previously we identified the mTORC2 specific subunit Sin1 as a direct binding partner for AGC kinases Akt and PKC. Sin1 mutants, which retain the ability to bind Rictor and mTOR, but fail to recruit their AGC client kinases, inhibit AKT and PKC priming and block cell growth. In this study, we demonstrate that uncoupling mTORC2 from AGC kinases in DLD1 colon cancer cells inhibits Akt activation and blocks tumour growth in vivo. Further we demonstrate, using time resolved two-site amplified FRET (A-FRET) analysis of xenograft tumours, that inhibition of tumour growth correlates with the degree of mTORC2 uncoupling from its downstream targets, as demonstrated for Akt. These data add weight to the body of evidence that mTORC2 represents a pharmacological target in cancer independently of mTORC1.

PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and ß) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.

Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion.

In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids.

Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.

Pseudokinase drug intervention: a potentially poisoned chalice.

Pseudokinases, the catalytically impaired component of the kinome, have recently been found to share more properties with active kinases than previously thought. In many pseudokinases, ATP binding and even some activity is preserved, highlighting these proteins as potential drug targets. In both active kinases and pseudokinases, binding of ATP or drugs in the nucleotide-binding pocket can stabilize specific conformations required for activity and protein-protein interactions. We discuss the implications of locking particular conformations in a selection of (pseudo)kinases and the dual potential impact on the druggability of these proteins.

mTORC2 targets AGC kinases through Sin1-dependent recruitment.

The protein kinase TOR (target of rapamycin) is a key regulator of cell growth and metabolism with significant clinical relevance. In mammals, TOR signals through two distinct multi-protein complexes, mTORC1 and mTORC2 (mammalian TOR complex 1 and 2 respectively), the subunits of which appear to define the operational pathways. Rapamycin selectively targets mTORC1 function, and the emergence of specific ATP-competitive kinase inhibitors has enabled assessment of dual mTORC1 and mTORC2 blockade. Little is known, however, of the molecular action of mTORC2 components or the relative importance of targeting this pathway. In the present study, we have identified the mTORC2 subunit Sin1 as a direct binding partner of the PKC (protein kinase C) ε kinase domain and map the interaction to the central highly conserved region of Sin1. Exploiting the conformational dependence for PKC phosphorylation, we demonstrate that mTORC2 is essential for acute priming of PKC. Inducible expression of Sin1 mutants, lacking the PKC-interaction domain, displaces endogenous Sin1 from mTORC2 and disrupts PKC phosphorylation. PKB (protein kinase B)/Akt phosphorylation is also suppressed by these Sin1 mutants, but not the mTORC1 substrate p70(S6K) (S6 kinase), providing evidence that Sin1 serves as a selectivity adaptor for the recruitment of mTORC2 targets. This inducible selective mTORC2 intervention is used to demonstrate a key role for mTORC2 in cell proliferation in three-dimensional culture.

Occupational hazards: allosteric regulation of protein kinases through the nucleotide-binding pocket.

Targeting the protein kinase ATP-binding pocket provides a significant opportunity for the treatment of disease. Recent studies have revealed a central activity-independent role for nucleotide pocket occupation in the allosteric behaviour of diverse kinases. Regulation of nucleotide pocket conformation with either nucleotides or ATP competitive inhibitors has revealed an added dimension to the targeting of kinases. In the present paper, using PKC (protein kinase C) as a paradigm, the liabilities and opportunities associated with the occupation of the nucleotide pocket are explored.

PKC and the control of localized signal dynamics.

Networks of signal transducers determine the conversion of environmental cues into cellular actions. Among the main players in these networks are protein kinases, which can acutely and reversibly modify protein functions to influence cellular events. One group of kinases, the protein kinase C (PKC) family, have been increasingly implicated in the organization of signal propagation, particularly in the spatial distribution of signals. Examples of where and how various PKC isoforms direct this tier of signal organization are becoming more evident.

PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity.

The protein kinase C (PKC) Ser/Thr kinases account for approximately 2% of the human kinome and regulate diverse cellular behaviors. PKC catalytic activity requires priming phosphorylations at three conserved sites within the kinase domain. Here we demonstrate that priming of PKC is dependent on the conformation of the nucleotide binding pocket but not on its intrinsic kinase activity. Inactive ATP binding site mutants are unprimed, but they become phosphorylated upon occupancy of the ATP binding pocket with inhibitors of PKC. We have exploited this property to screen for PKC inhibitors in vivo. Further, we generated a distinct class of kinase-inactive mutants that maintain the integrity of the ATP binding pocket; such mutants are constitutively primed and functionally distinct from ATP binding site mutants. These data demonstrate that autophosphorylation is not required for PKC priming and show how ATP pocket occupation can enable a kinase to mature as well as function.

Statins inhibit NK cell cytotoxicity by membrane raft depletion rather than inhibition of isoprenylation.

To investigate the potential determinants of the pleiotropic effects of statins, we measured NK cell cytotoxicity in samples from normal subjects and patients, including patients receiving statin therapy. In a multivariate analysis, NK cell cytotoxicity was related to total plasma cholesterol concentration rather than statin use. In vitro, we investigated the role of lipid modification, specifically the effects on membrane rafts and raft-dependent signal transduction. We demonstrate that statins reduce NK cell cytotoxicity and that membrane cholesterol depletion by cyclodextrins has a similar effect. In contrast, isoprenyl transferase inhibitors had little or no effect on NK cell function. We hypothesise that the pleiotropic effects of statins reflect changes in membrane cholesterol and, specifically, the density of membrane rafts. Moreover, there is likely to be a relationship between membrane cholesterol, membrane rafts and cell function that may be involved in the pathogenesis of cardiovascular and metabolic diseases.

Simvastatin inhibits lymphocyte function in normal subjects and patients with cardiovascular disease.

HMG-CoA reductase inhibitors (statins) block the mevalonate pathway, preventing biosynthesis of cholesterol and isoprenoids. We investigated the effect of simvastatin on lymphocytes from normal human subjects and cardiovascular disease patients in order to provide a model for the in vivo actions of statins. Thirteen healthy volunteers were treated with 40 mg per day of simvastatin following which mean total cholesterol was reduced by 23% (S.D.+/- 11.7%) and mean LDL-cholesterol by 36% (S.D.+/- 16.3%). Lymphocyte lipid raft levels, represented by Lyn and Fyn, were also reduced by simvastatin. Treatment with simvastatin did not alter ex vivo T-lymphocyte proliferation. However, the in vitro addition of 1 microM simvastatin reduced T-lymphocyte proliferation by 39% (S.D.+/- 18.1%) and a combination of prenyl transferase inhibitors reduced proliferation by 19% (S.D.+/- 22.7%). We also assessed the cytotoxicity of natural killer (NK) cells-a T-lymphocyte subset. NK cell cytotoxicity ex vivo was reduced by 30% (S.D.+/- 33.6%) following oral simvastatin treatment and by 56% (S.D.+/- 24.68%) after the in vitro addition of 1 microM simvastatin. Significant ex vivo reductions in T-cell proliferation and NK cell cytotoxicity were observed in patients with cardiovascular disease on treatment with statins. NK cells have been implicated in the pathogenesis of atherosclerosis, so the effect of statin therapy on NK cell cytotoxicity may contribute to the benefits of statins in cardiovascular disease.

Fluvastatin inhibits raft dependent Fcgamma receptor signalling in human monocytes.

Statins inhibit HMG-CoA reductase and thus block cholesterol and isoprenoid biosynthesis. Since statins also have anti-inflammatory effects, we investigated the effect of fluvastatin on monocyte Fcgamma receptor function. Fluvastatin (0.5-20 microM) inhibited Fcgamma receptor signal transduction at the level of tyrosine kinase activation, in a time and dose dependent manner. Initiation of tyrosine phosphorylation is not thought to involve prenylated proteins; thus, we hypothesised that fluvastatin might disrupt cholesterol and sphingolipid membrane rafts to impair signalling. Consistent with this hypothesis, fluvastatin decreased (and mevalonate rescued) signalling molecules within membrane rafts in parallel with effects on tyrosine phosphorylation events. Raft integrity was unaffected by prenyl transferase inhibitors. In addition, Fcgamma receptor mediated immune complex trafficking, activation of MAP kinases (ERK and p38), and downstream inflammatory mediator release (MMP-1 and IL-6) were blocked by fluvastatin. Thus, HMG-CoA reductase inhibition alters immune receptor signalling by disrupting membrane rafts essential for the initiation of signal transduction. Inhibition of Fcgamma receptor function may limit development and progression of atherosclerosis by decreasing monocyte/macrophage inflammatory mediator release. Since many receptors utilise cholesterol rich rafts this mechanism may have broader significance given the pleiotropic effects of statins.